Fig. 4

Concordance between LipoGlo electrophoresis and classical apolipoprotein B-containing lipoprotein (ApoB-LP) size characterization techniques. Density gradient ultracentrifugation (DGUC) was performed on pooled larval homogenate (4 days post fertilization (dpf)) from wild-type (WT), mtp^−/−, and apoC2^−/−, and separated into 10 equal fractions by drip elution (dense bottom fractions eluted first). a Fractions 2–10 were subjected to Native-polyacrylamide gel electrophoresis (Native-PAGE), and denser fractions showed higher electrophoretic mobility. Some fractions show a faint lower mobility band (indicated at right by white arrowhead), possibly indicative of lipoprotein dimerization. b A plate-based assays of NanoLuc activity revealed the expected enrichment of VLDL in apoC2^−/− mutants, and enrichment of LDL in mtp^−/− mutants (confirming results reported in Fig. 3b). c A refractometer (Bausch and Lomb) was used to determine the refractive index of each fraction and density was calculated via the formula D = 3.3508 × RI − 3.4675. DGUC showed highly reproducible density profiles between replicates and genotypes. d The density of WT fractions 4–9 was plotted as a function of peak electrophoretic mobility for that fraction, and the second-order polynomial function (y = 0.0796 × 2 − 0.1886x + 1.136) was able to represent this relationship with remarkable accuracy (R² = 0.97737), indicating that electrophoretic mobility is a useful proxy for lipoprotein density. e Fractions 4, 7, and 10 were subjected to negative-staining electron microscopy to directly visualize the size of particles in each fraction. Letters denote significantly different statistical groups by Games-Howell post hoc test. In the wild-type samples, the average particle diameter was 24.7 ± 5.6, 29.0 ± 4.1, and 34.9 ± 4.7 nm for fractions 4, 7, and 10, respectively. There was no significant difference in particle size between fraction 4 of the WT and mtp^−/− mutant samples (average diameter of 23.2 ± 6.6 nm). Particles were nearly undetectable in fractions 7 and 10 in the mtp^−/− mutant sample so particle diameter shows enormous variability. ApoB-LPs in each apocC2^−/− mutant fraction were significantly larger than all WT fractions, with diameters of 39.0 ± 8.0, 40.9 ± 7.2, and 39.1 ± 5.9 nm respectively (degrees of freedom (DF) = 8, n ≈ 170, Welch’s analysis of variance (ANOVA) p < 0.0001, Games–Howell p < 0.0001). f Representative images of lipoproteins from the three wild-type fractions are shown. g The second-order polynomial function y = 10.82 × 2 − 44.551x + 70.125 approximated the relationship between electrophoretic mobility and density in wild-type samples with reasonable accuracy (R² = 0.79978). Results represent pooled data from four independent experiments. Error bars denote standard deviation. Source data are provided as a Source Data file